Currently, biomolecular dynamics and stability are investigated predominantly in vitro, on the basis of which findings are extrapolated to explain function in the living cell. The dynamics of biomolecules in vitro are often explained in terms of energy landscapes, usually considered a property of the individual biomolecule, as described by Frauenfelder et al., The Energy Landscapes and Motions of Proteins, 254 Science 1598-1603 (1991), which is incorporated herein by reference. In the living cell, the biomolecular energy landscape is modulated by myriad interactions. The dynamics and stability of a biomolecule contained within a cell, where it is crowded by up to 400 mg of macromolecules per ml of cytosol, can differ substantially from the dynamics of the isolated biomolecule. Crowding also modifies the properties of cellular water, which, in turn, can couple back to influence the dynamics of biomolecules. Membranes and other large scale structures within the cell can also crowd or confine biomolecules, as can the active interaction with cellular transport machinery or chaperones.
Because of the advantages of studying proteins and other biomolecules within the living cell, several “in-cell” methods have emerged within the last two decades, yielding a variety of types of information. CARS (coherent anti-Stokes Raman scattering) microscopy reports on small molecule distributions inside living cells. FlAsH (fluorescein arsenical hairpin)-labeling can reveal slow urea-induced unfolding of proteins in bacterial cells. FRET (Förster resonant energy transfer) coupled with fluorescence microscopy can localize proteins, monitor protein-protein interactions and the motion of larger protein machinery. Fluorescent tracers coupled to FRAP (fluorescence recovery after photobleaching) or FCS (fluorescence correlation spectroscopy) can monitor diffusion processes on a micrometer length scale. NMR spectroscopy can reveal much detailed information about protein structure and dynamics inside living cells, but unlike the previously enumerated “single-cell” techniques, NMR requires multiple cells to take up isotope enriched proteins to enable detection of the desired protein. In-cell NMR experiments have been successful in yeast, E. Coli and mammalian cells.
Additionally, fluorescent tracers used in fluorescence recovery after photobleaching or fluorescence correlation spectroscopy experiments can be used to monitor diffusion processes on a micrometer length scale.